Method of classifying counting leucocytes

ABSTRACT

There is provided a method for classifying and counting leukocytes with abnormal DNA amount, which comprises: (1) a step of staining cells in a sample obtained from a hematological sample by treatment with a hemolytic agent to lyse erythrocytes, with a fluorescent dye which can make a difference in the fluorescence intensity at least among mature leukocytes, leukocytes with abnormal DNA amount and immature leukocytes; (2) a step of introducing the sample containing the stained cells into a flow cytometer to measure scattered light and fluorescence of the respective cells; (3) a step of classifying leukocytes and coincidence cells/platelet clumps utilizing a difference in the intensity of a scattered light peak and a difference in the scattered light width; (4) a step of classifying and counting mature leukocytes, leukocytes with abnormal DNA amount and immature leukocytes, utilizing a difference in the scattered light intensity and a difference in the fluorescence intensity of leukocytes classified in the step (3).

FIELD OF THE INVENTION

The present invention relates to a method for classifying and countingleukocytes. More particularly, it relates to a method for classifyingand counting leukocytes, particularly leukocytes with abnormal DNAamount, in hematological samples utilizing a flow cytometer.

BACKGROUND ART

In the field of clinical laboratory test, classifying and countingleukocytes with abnormal DNA amount or immature leukocytes provide veryuseful information in diagnosis of diseases. For example, leukocytes innormal peripheral blood generally consist of these five-types, i.e.,lymphocytes, monocytes, basophiles, eosinophils and neutrophils, and theDNA amount of leukocytes is constant. However, leukocytes with abnormalDNA amount appear, for example, in blood disorders such as viraldiseases or lymphocytic leukemia, and immature leukocytes appear in suchblood disorders as myclocytic leukemia.

Usually, in order to measure leukocytes with abnormal DNA amount, it isnecessary to mix and stain mononuclear leukocytes (lymphocytes andmonocytes), which are separated from leukocytes by specific gravitycentrifugation, or peripheral blood with a nucleic acid-staining dye anda hemolytic agent, and subsequently to carry out the measurement.Separation of the mononuclear leukocytes by specific gravitycentrifugation is complicated in operation and requires 1 hour or morefor completion of the separation. In addition, 30 minutes or more arerequired for mixing and staining mononuclear leukocytes or peripheralblood with the nucleic acid-staining dye and the hemolytic agent.

In general, in order to classify and count immature leukocytes, a bloodsmear is prepared, properly stained and observed under a microscope forclassification and count. On the other hand, in recent year, a varietyof full-automatic hematocytometers have been provided utilizing theprinciple of a flow cyctometer. Although normal leukocytes can be highlyprecisely classified and counted with these cytometers, immatureleukocytes can not precisely be detected and classified because thecytometers are greatly influenced by platelet clumps and coincidencecells.

On the other hand, it has been reported that immature leukocytes andnormal leukocytes can be simultaneously classified and counted bymaintaining the immature leukocytes in a viable state and treating otherleukocytes with a hemolytic agent which damages other leukocytes;staining the damaged cells with a fluorescent dye which can stain thedamaged cells; and measuring scattered light and fluorescence of theresulting blood corpuscles (Japanese Unexamined Patent Application Hei10(1998)-206423). In this method, however, it was not possible todifferentiate leukocytes with abnormal DNA amount from platelet clumpsand coincidence cells, thus it was difficult to precisely classify andcount leukocytes with abnormal DNA amount.

Thus, there is a need for a rapid, simple and highly precise method formeasuring leukocytes with abnormal. DNA amount and simultaneously,immature leukocytes.

DISCLOSURE OF THE INVENTION

According to the present invention, there is provided a method whichmakes it possible to classify and count leukocytes with abnormal DNAamount and, simultaneously, immature leukocytes and mature leukocytes,by maintaining immature leukocytes in a viable state, treating otherleukocytes with a hemolytic agent which damages other leukocytes,staining the damaged cells with a fluorescent dye which can staindamaged cells, measuring scattered light and fluorescence of theresulting blood corpuscles, and using the difference in the intensity ofthe scattered light peak and the difference in the scattered lightwidth.

Therefore, according to the invention, there is provided a method forclassifying and counting leukocytes, which comprises:

-   -   (1) a step of staining cells in a sample obtained from a        hematological sample by treatment with a hemolytic agent, with a        fluorescent dye which can make a difference in the fluorescence        intensity at least among mature leukocytes, leukocytes with        abnormal DNA amount and immature leukocytes;    -   (2) a step of introducing the sample containing the stained        cells into a flow cytometer to measure scattered light and        fluorescence of the respective cells;    -   (3) a step of classifying leukocytes and coincidence        cells/platelet clumps utilizing a difference in the intensity of        a scattered light peak and a difference in the scattered light        width;    -   (4) a step of classifying and counting mature leukocytes,        leukocytes with abnormal DNA amount and immature leukocytes,        utilizing a difference in the scattered light intensity and a        difference in the fluorescence intensity of leukocytes        classified in the step (3).

The term “hematological sample” as used herein refers to a body fluidsample containing leukocytes such as samples collected from peripheralblood, myelic needling fluid, urine, and the like.

The term “mature leukocytes” as used herein refers to maturelymphocytes, monocytes, and granulocytes.

The term “leukocytes with abnormal DNA amount” refers to leukocytes inwhich the amount of DNA is higher or lower than that in normalleukocytes. In the present invention, however, leukocytes with abnormalDNA amount mean leukocytes in which the amount of DNA is higher thanthat in the normal one.

The term “immature leukocytes” as used herein refers to immatureleukocytes which exist usually in bone marrow and do not occur in theperipheral blood. These include, for example, myeloblasts,promyelocytes, myelocytes, metamyelocytes, and the like. In some cases,promyelocytes, myelocytes, and metamyelocytes are collectively calledimmature corpuscles of granulocytic series. In addition, hemopoieticprecursor cells of leukocytic series such as myeloid series stem cells(CFU-GEMN), neutrophil-macrophage colony-forming cells (CFU-GM),eosinophil colony-forming cells (CFU-EOS), and the like, all of whichare cells in the differentiation stage prior to blast cells are includedin the scope of the immature leukocytes according to the invention.

The term “platelet clumps” as used herein refers to the one obtained byaggregation of two or more platelets.

The term “coincidence cells” as used herein refers to the state in whichtwo or more cells pass at approximately the same time through thedetection section of a flow cell and are counted as one cell.

The term “scattered light peak” as used herein refers to a peak ofsignal wave form obtained from scattered light, and the “scattered lightwidth” refers to the width of signal wave form obtained from scatteredlight.

In present the invention, a hematological sample is treated with ahemolytic agent to lyse erythrocytes. On the other hand, by thistreatment, immature leukocytes are not lysed or damaged, but matureleukocytes and leukocytes with abnormal DNA amount are damaged. When ahemolytic agent of a particular composition is allowed to act on cells,although the action mechanism is not clear, a part of the cell membranelipid component of particular cells is extracted (withdrawn) to make asmall hole on the cell membrane, through which a specific substance canpass. This is called damage. As a result, a dye molecule can penetrateinto the particular cells to stain the same. Therefore, the damagedmature leukocytes and leukocytes with abnormal DNA amount are in a statesuitable for staining. To the contrary, the undamaged immatureleukocytes are not stained by the dye because no hole is made throughwhich the dye is allowed to pass. Additionally, in the mature leukocytesand leukocytes with abnormal DNA amount, the amount of the dye to bebound depends on the amount of DNA contained in each cell. Therefore,when such cells are stained with the dye, the amount of the dye involvedin staining is varied depending on the DNA amount, resulting in adifference in the intensity of fluorescence from the stained cells. Forexample, in case of leukocytes with abnormal DNA amount which containtwice higher amount of DNA compared to mature leukocytes, the amount ofthe dye to be bound is twice higher than the normal mature leukocytes,thus these cells generate stronger fluorescence than the matureleukocytes. As a result, there can be produced a difference in theintensity of fluorescence among mature leukocytes, leukocytes withabnormal DNA amount and immature leukocytes.

The hemolytic agent used in the step (1) of the invention preferablycomprises a surfactant, a solubilizing agent, amino acid(s), and abuffer.

As a surfactant, a variety of agents can be employed, and preferred is apolyoxyethylene nonionic surfactant. Specifically, it includes thosehaving the following formula (II):R^(1II)—R^(2II)—(CH₂CH₂O)n_(II)-H   (II)(wherein R^(1II) represents a C₉₋₂₅ alkyl, alkenyl or alkynyl group;R^(2II) represents —O—,

or —COO—; and n_(II) is 10-40).

The C₉₋₂₅ alkyl group includes nonyl, decyl, undecyl, dodecyl, tridecyl,tetradecyl, and the like. The C₉₋₂₅ alkenyl group includes dodecenyl,tetradecenyl, and the like. The C₉₋₂₅ alknyl group includes dodecynyl,undecynyl, dodecynyl, and the like.

More specifically, polyoxyethylene(20)lauryl ether,polyoxyethylene(15)oleyl ether, and polyoxyethylene(16)oleyl ether arepreferred.

The surfactant may be used in the form of aqueous solution. For example,the concentration of the polyoxyethylene nonionic surfactant in waterdepends on the type of the surfactant used, however, may be in the rangeof 0.1-2.0 g/L (preferably, 0.5-1.5 g/L) for polyoxyethylene(20)laurylether, 1-9 g/L (preferably, 3-7 g/L) for polyoxyethylene(15)oleyl ether,and 5-50 g/L (preferably, 15-35 g/L) for polyoxyethylene(16)oleyl ether.The polyoxyethylene nonionic surfactant, when the carbon number of thehydrophobic group is the same, shows a more potent cell-damagingproperty with decrease of the number of nil, and this potency decreaseswith increase of the number of nil. In addition, when the number of nilis the same, the cell-damaging potency increases with decrease of thecarbon number of the hydrophobic group. In consideration of this pointof view, the concentration required for a surfactant may easily bedetermined by an experiment using the above-mentioned values asstandards.

The solubilizing agent is used to give damage to the cell membrane ofblood. corpuscles and reduce their size. Specifically, one or moreselected from the followings:

-   -   sarcosine derivatives of the formula (III):    -   (wherein R1III is a C₁₀₋₂₂ alkyl group; and n_(III) is 1-5) or        salts thereof;    -   cholic acid derivatives of the formula (IV):    -   (wherein R^(1IV) is a hydrogen atom or a hydroxy group);    -   and    -   methylglucanamides of the formula (V):    -   (wherein n^(V) is 5-7)    -   may be used.

The C₁₀₋₂₂ alkyl group includes decyl, dodecyl, tetradecyl, oleyl, andthe like.

Specifically, sodium N-lauroylsarcosinate, sodium lauroyl methylβ-alanine, lauroylsarcosine, CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), CHAPSO(3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate),MEGA8 (octanoyl-N-methylglucamide), MEGA9 (nonanoyl-N-methylglucamide),MEGA10 (decanoyl-N-methylglucamide), and the like are preferably used.

The concentration of the solubilizing agent is preferably 0.2-2.0 g/Lfor sarcosine acid derivatives or their salts, 0.1-0.5 g/L for cholicacid derivatives, and 1.0-8.0 g/L for methylglucanamide.

In addition, n-octyl β-glucoside, sucrose monocaprate,N-formylmethylleucylalanine and the like can be used as solubilizingagents, which may preferably be used in a concentration of 0.01-50.0g/L.

The amino acid is used to immobilize the cytoplasm and cell membrane ofimmature leukocytes. For example, it is possible to use amino acidswhich constitute proteins, preferably glutamic acid, valine,particularly sulfur-containing amino acids such as methionine, cystineand cysteine are used; and most preferred is methionine. The amino acidmay be used in the range of 1-50 g/L; preferably 8-12 g/L for glutamicacid, and 16-24 g/L for methionine.

As for the buffer, Good buffer such as HEPES or phosphate buffer maypreferably be added with a pH adjusting agent such as sodium hydroxide,and if necessary, with an osmotic pressure adjusting agent such assodium chloride to obtain pH of 5.0-9.0 and an osmotic pressure of150-600 mOsm/kg.

As for the hemolytic agent according to the present invention, it ispreferable to use the hemolytic agent described in Japanese UnexaminedPatent Publication No. Hei 6(1994)-273413 which comprises (1) apolyoxyethylene nonionic surfactant; (2) a solubilizing agent to givedamage to cell membrane of blood corpuscles and reduce their size; (3)an amino acid; and (4) a buffer by which pH and osmotic pressure of theliquid are adjusted to 5.0-9.0 and 150-600 mOsm/kg, respectively, and abuffer by which the conductivity is adjusted to 6.0-9.0 mS/cm.

The fluorescent dye according to the invention which can make adifference in the fluorescence intensity among mature leukocytes,leukocytes with abnormal DNA amount and immature leukocytes may be anykind as far as it can stain either of damaged cells or immatureleukocytes. The dye which can stain damaged cells is preferred. Such adye can stain all of the cells including blood corpuscles in the sample.

The dye which can stain damaged cells includes dyes which havespecificity to cell nuclei, particularly DNA, or dyes which havespecificity to RNA. For this purpose, some cationic dyes are preferablyused.

In general, the cationic dye passes through a cell membrane of a viablecell to stain the intracellular component. It has been well known,however, that certain cationic dyes (for example, ethidium bromide,propidium iodide, etc.) do not pass through viable cells and can stainonly damaged cells.

Specifically, the fluorescence dye includes the above-mentioned ethidiumbromide and propidium iodide as well as ethidium-acridine heterodimer(commercially available from Molecular Probes), ethidium azide, ethidiumhomodimer-1, ethidium homodimer-2, ethidium monoazide, TOTO-1, TO-PRO-1,TOTO-3, and TO-PRO-3. When a He—Ne or red semiconductor laser is used asa light source, a dye represented by the formula (I):

(wherein R^(1I) is a hydrogen atom or a lower alkyl group; R^(2I) andR^(3I) each is a hydrogen atom, a lower alkyl group or a lower alkoxygroup; R^(4I) is a hydrogen atom, an acyl group or a lower alkyl group;R^(5I) is a hydrogen atom or a lower allyl group which may besubstituted; Z is sulfur atom, oxygen atom, or carbon atom which issubstituted by a lower alkyl group; n^(I) is 1 or 2; and X^(I −) is ananion) may be used as a suitable dye.

The lower alkyl group with regard to R^(1I) of the above formula means aC₁₋₆ straight or branched alkyl group. It includes methyl, ethyl,propyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl groupsand particularly, methyl and ethyl groups are preferred.

The lower alkyl group with regard to R^(2I) and R^(3I) includes the samegroups as the above; and the lower alkoxy group means a C₁₋₆ alkoxygroup and includes, for example methoxy, ethoxy, propoxy, and the like;and particularly methoxy and ethoxy groups are preferred.

The acyl group with regard to R^(4I) is preferably an acyl group derivedfrom an aliphatic carboxylic acid and includes, for example, acetyl,propionyl, and the like; and particularly, acetyl group is preferred.The lower alkyl group includes the same groups as the above.

The lower alkyl group with regard to R^(5I) includes the same groups asthe above; the lower alkyl group which may optionally be substitutedmeans the lower alkyl group which may be substituted by 1 to 3 hydroxygroup(s), halogen atom (fluorine, chlorine, bromine or iodine) and thelike; and particularly, methyl and ethyl groups substituted by onehydroxy group are preferred.

The lower alkyl group with regard to Z includes the same groups as theabove; and as Z, a sulfur atom is preferred.

The anion with regard to X^(I−) includes halogen ions (fluorine,chlorine, bromine or iodine ion), boron halide ions (BF⁴⁻, BCl⁴⁻, BBr⁴⁻,etc.), phosphorus compound ions, halogeno-oxygen acid ions,fluorosulfuric acid ions, methyl sulfate ions, and tetraphenyl boroncompound ions which have a halogen or halogeno-alkyl group as asubstituent on the aromatic rings. Particularly, bromine ion or BF⁴⁻ ispreferred.

The above-mentioned dyes may be used alone or in combination of two ormore species. A specific example of the above-mentioned dyes preferablyincludes those as described below, without limiting the presentinvention.

In a preferred embodiment of the step (1), a solution containing thehemolytic agent and the fluorescence dye is mixed with a hematologicalsample. Alternatively, the fluorescence dye may be dissolved, inadvance, to an aqueous organic solvent such as ethylene glycol and mixedwith the hemolytic agent upon use. This case is preferable because thestorage stability of the dye can be improved. The concentration of thedye may suitably be decided depending on the species of the dye to beused. For example, ethidium bromide may be used in the range of 0.01-100mg/L, preferably 0.1-30 mg/L.

Mixing of the hematological sample with the hemolytic agent containingthe fluorescence dye may preferably be carried out at a ratio ofhematological sample: hemolytic agent containing a fluorescencedye=1:10-1:1000, at a temperature of 20-40° C. for a reaction period of5 seconds to 5 minutes. When the reaction temperature is high, it ispreferred to reduce the reaction time. In the invention, the measurementof the amount of DNA in a sample containing immature leukocytes can beeffected by extending the reaction time in order to stain immatureleukocytes. In this case, the reaction time is preferably in a periodof, for example, 10 seconds to 5 minutes.

In the step (2), thus prepared sample for measurement is introduced intoa flow cytometer, in which scattered light and fluorescence of therespective stained cells in the sample are measured.

FIG. 18 shows an oblique perspective figure illustrating an opticalsystem of flow cytometer which can be used in the invention. In thisfigure, a beam emitted from a laser 21 irradiates an orifice portion ofa sheath flow cell 23 through a collimator lens 22. The forwardscattered light emitted by the blood corpuscles passing through theorifice portion is introduced into a photodiode 26 through a condensinglens 24 and a pin hole plate 25.

On the other hand, with regard to side scattered light and sidefluorescence emitted by the blood corpuscles passing through the orificeportion, the side scattered light is introduced into a photomultipliertube (hereinafter referred to as “photomul”) 29 through a condensinglens 27 and a dichroic mirror 28, and the side fluorescence isintroduced into a photomul 31 through the condensing lens 27, thedichroic mirror 28, a filter 29 and a pin hole plate 30.

The forward scattered light signal generated by the photodiode 26, theside scattered light signal generated by the photomul 29, and the sidefluorescence signal generated by the photomul 31 are amplified byamplifiers 32, 33, and 34, respectively, and input an analyzing part 35.

The term “scattered light” as used herein refers to scattered lightwhich can be measured with a commercially available flow cytometer, andincludes forward low angle scattered light (for example, receiving angleless than 0-5°), forward high angle scattered light (for example,receiving angle around 5-20°), and side scattered light, and preferablyforward low angle scattered light and, as further scattered light, sidescattered light are chosen. The side scattered light reflectsintracellular information such as a nuclear form.

As for fluorescence, a suitable receiving wavelength is selectedaccording to the dye used. The fluorescent signals reflect chemicalproperties of cells.

The light source of flow cytometer is not particularly limited, and isselected from the sources having a suitable wavelength for excitation ofthe dye. For example, argon laser, He—Ne laser, red semiconductor laser,blue semiconductor laser, and the like may be used. In particular, asemiconductor laser is much cheaper than gas laser, thus cost for anapparatus can be reduced drastically.

In the step (3), platelet clumps/coincidence cells and leukocytes areclassified using the difference in the intensity of the scattered lightpeak and the difference in the scattered light width. Specifically, forexample, a scattergram is prepared in which the X-axis indicates theforward scattered light width and the Y-axis indicates the forwardscattered light peak. As shown in FIG. 1, for example, platelet clumpsand coincidence cells as well as leukocytes and ghost form respectivegroups which distribute in the scattergram. From all of the cells on thescattergram, the platelet clumps and coincidence cells are removed toclassify the platelet clumps/coincidence cells and the leukocytes/ghost.By this operation, it is possible to prevent an appearance of plateletclumps and coincidence cells in the area of the leukocytes with abnormalDNA amount, and to precisely classify and count leukocytes with abnormalDNA amount.

Subsequently, in the step (4), mature leukocytes, leukocytes withabnormal DNA amount and immature leukocytes are classified and countedutilizing the difference in the scattered light intensity, and thedifference in the fluorescence intensity of the components classified inthe step (3). Specifically, for example, a scattergram, in which theX-axis indicates the fluorescence intensity and the Y-axis indicates theforward scattered light intensity, is prepared only with groups of theblood corpuscle components excluding the above-mentioned platelet clumpsand coincidence cells. As shown in FIG. 2, for example, respectivegroups of mature leukocytes, leukocytes with abnormal DNA amount andimmature leukocytes as well as the group formed of erythrocyte ghost,respectively, are distributed in the scattergram. The areas of therespective groups are established using a suitable analyzing software,and the cells included in those areas are classified and counted. Thus,the number of mature leukocytes, the number of immature leukocytes andthe number of leukocytes with abnormal DNA amount can be obtained.

Further, in the step (5), the ratio of the mature leukocytes or immatureleukocytes relative to the leukocytes with abnormal DNA amount iscalculated from the number of leukocytes with abnormal DNA amount andthe number of mature leukocytes or the number of immature leukocytes.

Further, in the step (6), the ratio of the immature leukocytes relativeto the mature leukocytes is calculated from the number of matureleukocytes and the number of immature leukocytes.

Further, in the step (7), different kind of scattered light is furthermeasured in the step (2), and a scattergram is prepared usingdifferences of the scattered light intensity and fluorescence intensityof the mature leukocytes obtained in the step (4). For example, ascattergram as shown in FIG. 3, in which the X-axis indicates theintensity of red fluorescence and the Y-axis indicates the intensity ofside scattered light, is prepared. In the scattergram, at least 3species, for example, lymphocytes, monocytes and granulocytes formrespective groups, and are distributed. The areas of the respectivegroups are established using a suitable analyzing software, and thecells included in those areas are classified and counted. Thus,lymphocytes, monocytes and granulocytes can be classified and counted.

Further, in the step (8), different kind of scattered light is furthermeasured in the step (2), and a scattergrain is prepared usingdifferences of the scattered light intensity and fluorescence intensityof the immature leukocytes obtained in the step (4). For example, ascattergram as shown in FIG. 3, in which the X-axis indicates theintensity of red fluorescence and the Y-axis indicates the intensity ofside scattered light, is prepared. In the scattergram, at least 2species, for example, myeloblasts and immature corpuscles ofgranulocytic series form respective groups, and are distributed. Theareas of the respective groups are established using a suitableanalyzing software, and the cells included in those areas are classifiedand counted. Thus, myeloblasts and immature corpuscles of granulocyticseries can be classified and measured.

In this connection, the steps (7) and (8) may be carried out separatelyor simultaneously. When both steps are carried out simultaneously, it ispreferable to prepare a scattergram using the difference in thescattered light intensity and the difference in the fluorescenceintensity of the components being removed of ghost in the step (4),since a scattergram as shown in FIG. 3 is obtained and the matureleukocytes and immature leukocytes can be classified into furthermultiple groups and counted.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a conceptual illustration showing the positions at which therespective cells appear as classified and counted according to themethod of the invention.

FIG. 2 is a conceptual illustration showing the positions at which therespective cells appear as classified and counted according to themethod of the invention.

FIG. 3 is a conceptual illustration showing the positions at which therespective cells appear as classified and counted according to themethod of the invention.

FIG. 4 is a scattergram of Example 1 in which the X-axis indicates theforward low angle scattered light width and the Y-axis indicates theforward low angle scattered light peak.

FIG. 5 is a scattergram of Example 1 in which the X-axis indicates thered fluorescence intensity and the Y-axis indicates the forward lowangle scattered light intensity.

FIG. 6 is a scattergram of Example 1 in which the X-axis indicates theside scattered light intensity and the Y-axis indicates the redfluorescence intensity.

FIG. 7 represents results from the present method and from a standardmethod for measurement of the DNA amount.

FIG. 8 is a scattergram of Example 2 in which the X-axis indicates theforward low angle scattered light width and the Y-axis indicates theforward low angle scattered light peak.

FIG. 9 is a scattergram of Example 2 in which the X-axis indicates thered fluorescence intensity and the Y-axis indicates the forward lowangle scattered light intensity.

FIG. 10 is a scattergram of Example 2 in which the X-axis indicates theside scattered light intensity and the Y-axis indicates the redfluorescence intensity.

FIG. 11 is a scattergram of Example 3 depicted for 7 seconds of thereaction time in Example 3, in which the X-axis indicates the forwardlow angle scattered light width and the Y-axis indicates the forward lowangle scattered light peak.

FIG. 12 is a scattergram depicted for 7 seconds of the reaction time inExample 3, in which the X-axis indicates the red fluorescence intensityand the Y-axis indicates the forward low angle scattered lightintensity.

FIG. 13 is a scattergram depicted for 7 seconds of the reaction time inExample 3, in which the X-axis indicates the side scattered lightintensity and the Y-axis indicates the red fluorescence intensity.

FIG. 14 is a scattergram depicted for 13 seconds of the reaction time inExample 3, in which the X-axis indicates the forward low angle scatteredlight width and the Y-axis indicates the forward low angle scatteredlight peak.

FIG. 15 is a scattergram depicted for 13 seconds of the reaction time inExample 3, in which the X-axis indicates the red fluorescence intensityand the Y-axis indicates the forward low angle scattered lightintensity.

FIG. 16 is a scattergram depicted for 13 seconds of the reaction time inExample 3, in which the X-axis indicates the side scattered lightintensity and the Y-axis indicates the red fluorescence intensity.

FIG. 17 represents results from the present method, in which thereaction time is 13 seconds, and from a standard method for measurementof the DNA amount.

FIG. 18 is an oblique perspective figure showing an optical system offlow cytometer usable in the invention.

EMBODIMENTS OF THE INVENTION

The present invention will be explained by the following examples inmore detail. A variety of alteration and modification can be applied tothe invention, and accordingly, the scope of the invention is notlimited by the following examples.

EXAMPLE 1

A reagent comprising an aqueous solution of the following compositionwas prepared. (The present method) Polyoxyethylene(16)oleyl ether 24.0 gSodium N-lauroylsarcosinate 1.5 g DL-Methionine 20.0 g 1N-NaOH 0.3 gNaCl 4.0 g Dye of formula (VI) 3.0 mg HEPES 12.0 g Pure water 1000 ml

The above-mentioned reagent (1 ml) was mixed with 33 μl of bloodcollected from a patient suffering from lymphoid leukemia, and after alapse of 10 seconds, forward low angle scattered light, side scatteredlight, and red fluorescence were measured with a flow cytometer (lightsource: red semiconductor laser, wavelength: 633 nm). (A standard methodfor measurement of the DNA amount) Trisodium citrate 100 mg Triton X-100(Wako Pure Chemical Industries, Ltd.) 0.2 g Propidium iodide (Sigma) 0.2g RO water 100 ml

The above-mentioned reagent (1 ml) was mixed with 100μl of bloodcollected from the above-mentioned patient, and after a lapse of 30minutes, red fluorescence was measured with a flow cytometer (lightsource: argon ion laser, wavelength: 488 nm).

The results are shown in a scattergram (FIG. 4) in which the X-axisindicates the forward low angle scattered light width and the Y-axisindicates the forward low angle scattered light peak, a scattergram(FIG. 5) in which the X-axis indicates the red fluorescence intensityand the Y-axis indicates the forward low angle scattered lightintensity, and a scattergram (FIG. 6) in which the X-axis indicates theside scattered light intensity and the Y-axis indicates the redfluorescence intensity.

The above-mentioned blood was stained by May-Grünwald stain and observedvisually under a microscope. Leukocytes were classified intolymphocytes, monocytes and granulocytes. In addition, using theabove-mentioned blood, the DNA amount of leukocytes was determined by aflow cytometer according to the standard method for measurement of theDNA amount. The results are shown in Table 1 and FIG. 7. TABLE 1 Resultsby the present method and by the visual observation Present Visualmethod method Lymphocyte (%) 38.3 39.0 Monocyte (%) 4.5 3.0 Granulocyte(%) 47.7 49.0 Myeloblast (%) 2.3 1.5 Granulocytic series immaturecorpuscle (%) 2.1 1.5 Leukocyte with abnormal DNA amount (%) 5.1 6.0Mature leukocyte: Leukocyte with abnormal 17.7:1 15.2:1 DNA amountImmature leukocyte: Leukocyte with 0.86:1  0.5:1 abnormal DNA amountMature leukocyte: Immature leukocyte 20.6:1 30.3:1

From the above results, it was shown that by the method of theinvention, it is possible to measure leukocytes with abnormal DNA amountand immature leukocytes at the same time as the similar level as that ofvisual observation in a rapid, simple and highly precise way.

EXAMPLE 2

A reagent comprising an aqueous solution of the following compositionwas prepared. (The present method) Polyoxyethylene(16)oleyl ether 24.0 gSodium N-lauroylsarcosinate 1.5 g DL-Methionine 20.0 g 1N-NaOH 0.3 gNaCl 4.0 g Dye of formula (VII) 3.0 mg HEPES 12.0 g Pure water 1000 ml

The above-mentioned reagent (1 ml) was mixed with 33 μl of bloodcollected from a patient suffering from acute myelocytic leukemia (AML),and after a lapse of 10 seconds, forward low angle scattered light, sidescattered light, and red fluorescence were measured with a flowcytometer (light source: red semiconductor laser, wavelength: 633 nm).(A standard method for measurement of the DNA amount) Trisodium citrate100 mg Triton X-100 (Wako Pure Chemical Industries, Ltd.) 0.2 gPropidium iodide (Sigma) 0.2 g RO water 100 ml

The above-mentioned reagent (1 ml) was mixed with 100 μl of bloodcollected from the above-mentioned patient, and after a lapse of 30minutes, red fluorescence was measured with a flow cytometer (lightsource: argon ion laser, wavelength: 488 nm).

The results are shown in a scattergram (FIG. 8) in which the X-axisindicates the forward low angle scattered light width and the Y-axisindicates the forward low angle scattered light peak, a scattergram(FIG. 9) in which the X-axis indicates the red fluorescence intensityand the Y-axis indicates the forward low angle scattered lightintensity, and a scattergram (FIG. 10) in which the X-axis indicates theside scattered light intensity and the Y-axis indicates the redfluorescence intensity.

The above-mentioned blood was stained by May-Grünwald stain and observedvisually under a microscope. The leukocytes were classified intolymphocytes, monocytes and granulocytes. The results are shown in Table2. TABLE 2 Present Visual method method Lymphocyte (%) 3.6 4.0 Monocyte(%) 8.6 10.0 Granulocyte (%) 77.0 75.0 Myeloblast (%) 3.6 5.0Granulocytic immature corpuscle (%) 7.2 6.0 Leukocyte with abnormal DNAamount (%) 0.0 0.0 Mature leukocyte: Leukocyte with abnormal 89.2:089.0:0 DNA amount Immature leukocyte: Leukocyte with 10.8:0 11.0:0abnormal DNA amount Mature leukocyte: Immature leukocyte 8.26:1  8.1:1

From the above results, it was shown that by the method of theinvention, it is possible to measure leukocytes with abnormal DNA amountand immature leukocytes at the same time in a rapid, simple and highlyprecise way.

EXAMPLE 3

The reagents with the same composition as in Example 1 were used. Thereagent of the present method (1 ml) was mixed with 33 μl of bone marrowfluid collected from a patient suffering from osteomyelodysplasiasyndrome, and after a lapse of 7 seconds and 13 seconds, forward lowangle scattered light, side scattered light, and red fluorescence weremeasured with a flow cytometer (light source: red semiconductor laser,wavelength: 633 nm).

(A standard Method for Measurement of the DNA Amount)

The reagent (1 ml) for the standard method for measurement of the DNAamount was mixed with 100 μl of bone marrow fluid collected from theabove-mentioned patient, and after a lapse of 30 minutes, redfluorescence was measured with a flow cytometer (light source: argon ionlaser, wavelength: 4-88 nm).

The results are shown in a scattergram (FIG. 11) for the case of 7seconds of the reaction time in which the X-axis indicates the forwardlow angle scattered light width and the Y-axis indicates the forward lowangle scattered light peak, a scattergram (FIG. 12) for the case of 7seconds of the reaction time in which the X-axis indicates the redfluorescence intensity and the Y-axis indicates the forward low anglescattered light intensity, a scattergram (FIG. 13) for the case of 7seconds of the reaction time in which the X-axis indicates the sidescattered light intensity and the Y-axis indicates the red fluorescenceintensity, a scattergram (FIG. 14) for the case of 13 seconds of thereaction time in which the X-axis indicates the forward low anglescattered light width and the Y-axis indicates the forward low anglescattered light peak, a scattergram (FIG. 15) for the case of 13 secondsof the reaction time wherein the X-axis indicates the red fluorescenceintensity and the Y-axis indicates the forward low angle scattered lightintensity, and a scattergram (FIG. 16) for the case of 13 seconds of thereaction time in which the X-axis indicates the side scattered lightintensity and the Y-axis indicates the red fluorescence intensity.

The above-mentioned bone marrow fluid was stained by May-Grünwald stainand observed visually under a microscope. The leukocytes were classifiedinto lymphocytes, monocytes and granulocytes. In addition, using theabove-mentioned blood, the DNA amount of the leukocytes was determinedby a flow cytometer according to the standard method for measurement ofthe DNA amount. The results are shown in Table 3. Table 4 and FIG. 17show the results obtained by the method of the invention conducted inthe reaction time of 13 seconds and by the standard method formeasurement of the DNA amount. TABLE 3 Present method (Reaction Visualtime: 7 sec) method Lymphocyte (%) 9.5 8 Monocyte (%) 16.4 18Granulocyte (%) 6.8 9 Myeloblast (%) 2.3 1 Granulocytic immaturecorpuscle (%) 65.0 51 Leukocyte with abnormal DNA amount (%) 0 13 Matureleukocyte: Leukocyte with abnormal 32.7:0  2.7:1 DNA amount Immatureleukocyte: Leukocyte with 67.3:0   4:1 abnormal DNA amount Matureleukocyte: Immature leukocyte 0.49:1 0.67:1

TABLE 4 Standard method Present method for measuring DNA (Reaction time:13 sec) amount (%) (%) Leukocyte with abnormal 11 13 DNA amount Normalleukocyte (in 89 87 DNA amount)

In conclusion, mature leukocytes and immature leukocytes can be measuredprecisely when the reaction time is 7 seconds, and leukocytes withabnormal DNA amount contained in immature leukocytes can be detectedwhen the reaction time is 13 seconds.

As shown above, according to the invention it is possible to rapidly andsimply measure leukocytes in which the DNA amount is abnormal.

1. A method for classifying and counting leukocytes, which comprises:(1) a step of staining cells in a sample obtained from a hematologicalsample by treatment with a hemolytic agent, with a fluorescent dye whichcan make a difference in the fluorescence intensity at least amongmature leukocytes, leukocytes with abnormal DNA amount and immatureleukocytes; (2) a step of introducing the sample containing the stainedcells into a flow cytometer to measure scattered light and fluorescenceof the respective cells; (3) a step of classifying a first group and asecond group utilizing a difference in the intensity of a scatteredlight peak and a difference in the scattered light width, the firstgroup including leukocvtes and second group including coincidence cellsand platelet clumps; (4) a step of classifying and counting matureleukocytes, leukocytes with abnormal DNA amount and immature leukocytes,utilizing a difference in the scattered light intensity and a differencein the fluorescence intensity of leukocytes classified in the step (3).2. The method according to claim 1, which further comprises a step ofcalculating a ratio of mature leukocytes or immature leukocytes relativeto leukocytes with abnormal DNA amount from a number of leukocytes withabnormal DNA amount and a number of mature leukocytes or immatureleukocytes.
 3. The method according to claim 1, which further comprisesa step of calculating a ratio of immature leukocytes relative to matureleukocytes from a number of mature leukocytes and a number of immatureleukocytes.
 4. The method according to claim 1, which comprises a stepof further measuring a different kind of scattered light in the step(2), and classifying mature leukocytes into at least three groups andcounting the same using the difference in the scattered light intensityand the difference in the fluorescence intensity of mature leukocytesobtained in the step (4).
 5. The method according to claim 1, whichcomprises a step of further measuring a different kind of scatteredlight in the step (2), and classifying immature leukocytes into at leasetwo groups and counting the same using the difference in the scatteredlight intensity and the difference in the fluorescence intensity ofimmature leukocytes obtained in the step (4).
 6. The method according toclaim 1, wherein the fluorescent dye is selected from the groupconsisting of a compound represented by the formula (I):

(wherein R^(1I) is a hydrogen atom or a lower alkyl group; R^(2I) andR^(3I) each is a hydrogen atom, a lower alkyl group or a lower alkoxygroup; R^(4I) is a hydrogen atom, an acyl group or a lower alkyl group;R^(5I) is a hydrogen atom or a lower alkyl group which may besubstituted; Z is sulfur atom, oxygen atom, or carbon atom which issubstituted by a lower alkyl group; n^(I) is 1 or 2; and X^(I−) is ananion), ethidium bromide, propidium iodide, ethidium-acridineheterodimer, ethidium azide, ethidium homodimer-1, ethidium homodimer-2,ethidium monoazide, TOTO-1, TO-PRO-1, TOTO-3, and TO-PRO-3.
 7. Themethod according to claim 1, wherein the hemolytic agent comprises thefollowing components: (1) a polyoxyethylene nonionic surfactant; (2) asolubilizing agent to give damage to cell membrane of blood corpusclesand reduce their size; (3) an amino acid; and (4) a buffer by which pHand osmotic pressure of the liquid are adjusted to 5.0-9.0 and 150-600mOsm/kg, respectively.
 8. The method according to claim 7, wherein thepolyoxyethylene nonionic surfactant comprises a compound represented bythe following formula (II):R^(1II)—R^(2II)—(CH₂CH₂O)n_(II)-H   (II) (wherein R^(1II) represents aC₉₋₂₅ alkyl, alkenyl or alkynyl group; R^(2II) represents —O—,

or —COO—; and n_(II) is 10-40).
 9. The method according to claim 7,wherein the solubilizing agent is a compound selected from the groupconsisting of a sarcosine derivative of the formula (III):

(wherein R^(1II) is a C₁₀₋₂₂ alkyl group; and n^(III) is 1-5) or saltsthereof; a cholic acid derivative of the formula (IV):

(wherein R^(1IV) is a hydrogen atom or a hydroxy group); and amethylglucanamide of the formula (V):

(wherein n^(V) is 5-7).
 10. The method according to claim 1, whereinscattered light to be measured is selected from forward low anglescattered light, forward high angle scattered light and side scatteredlight.
 11. A system for classifying and counting leukocytes whichcomprises: a flow cytometer comprising an orifice portion in which asample for measurement passes through, a light source which irradiateslight to the orifice portion, a first light receiving portion whichreceives scattered light emitted from the orifice portion and a secondlight receiving portion which receives fluorescence emitted from theorifice portion, said sample for measurement being prepared by a step ofmixing a hematological sample with a hemolytic agent and a step ofstaining cells in the resulting mixture with a fluorescent dye which canmake a difference in the fluorescence intensity at least among matureleukocytes, leukocytes with abnormal DNA amount and immature leukocytes;and an analyzing part in which the sample for measurement is analyzed bya step of classifying a first group and a second group utilizing adifference in the intensity of a scattered light peak and a differencein the scattered light width, the first group including leukocytes andthe second group including coincidence cells and platelet clumps and astep of classifying and counting mature leukocytes, leukocytes withabnormal DNA amount and immature leukocytes, utilizing a difference inthe intensity and a difference in the fluorescence intensity ofleukocytes classified.